The combination of Hepatitis C virus (HCV) infection and alcohol consumption produces a more severe liver disease than either condition alone with increased rates of both cirrhosis and hepatocellular carcinoma. We have recently discovered that the combination of HCV and alcohol causes a loss in the activity of the transcription factor FOXO3, a member of the forkhead box family of transcription factors that is responsible for oxidant stress resistance, control of cell proliferation, apoptosis, and insulin sensitivity. FOXO3 is also an established tumor suppressor molecule. Its cellular localization and transcriptional activity are regulated by a series of post translational modifications (PTMs) of the protein. Our preliminary data has shown that either HCV infection alone or alcohol exposure alone increases FOXO3 activity, but the combination of HCV and alcohol results in acetylation of FOXO3 and loss of its transcriptional activity. We also observed decreased FOXO3 activity in liver biopsies from chronic hepatitis C patients and abnormally localized FOXO3 in 85% of HCV-associated hepatocellular carcinomas. The central hypothesis of this proposal is that HCV causes changes in FOXO3 phosphorylation, acetylation and ubiquitination. These PTMs result from altered activity of upstream kinases, acetyl transferases, deacetylases, ubiquitin conjugating enzymes and deubiquitinases. The HCV/alcohol changes in FOXO3 and are responsible for enhanced sensitivity to alcohol and the loss of tumor suppressor activity in hepatocellular carcinoma. Therapeutic approaches targeting FOXO3 have the potential to improve HCV-alcohol associated liver injury and prevent the development of hepatocellular carcinoma. We will examine this hypothesis by three specific aims. They are: (1) to develop a novel nanoscale isoelectic focusing method to determine the nature and mechanism of HCV/ethanol-induced posttranslational modifications of FOXO3; (2) to determine why there is abnormal cytosolic accumulation of FOXO3 in hepatocarcinogenesis and whether this affects tumor behavior; and (3) to examine the role of HCV-induced FOXO3 acetylation in alcohol associated hepatotoxicity and test whether augmenting FOXO3 activity has potential to reduce liver injury. Methods to be employed include a novel isoelectric focusing assay for FOXO3 PTMs, examination of liver explants samples from patients undergoing liver transplantation for hepatocellular carcinoma, and cellular and mouse models of HCV-alcohol interactions. The hypotheses and approaches in this study are highly novel. Successful completion of these studies will identify the molecular changes in FOXO3 that result specifically from the HCV-alcohol interaction, determine how these contribute to cirrhosis and liver carcinogenesis, and use this information to establish the foundation for using FOXO3 manipulation as a therapeutic measure to improve outcome of patients with Hepatitis C and alcohol consumption.